Background: Giardia duodenalis is a common parasitic diarrheal agent in humans, especially in developing countries. The aim of this study was to investigate the prevalence and multilocus genetic characterization of G. duodenalis in patients with diarrhea and animals in northeastern China, and to assess the epidemiological role of animals in the transmission of human giardiasis. Methods: A total of 1739 fecal specimens from 413 diarrheal patients and 1326 animals comprising 16 mammal species were collected in Heilongjiang Province of China and screened for G. duodenalis by PCR and sequencing of the SSU rRNA gene. All G. duodenalis-positive specimens were subtyped by PCR and sequencing of the bg, tpi, and gdh genes. To detect additional mixed infections of different assemblages, assemblage A/B/E-specific PCRs were performed to amplify the tpi gene. Results: Sequence analysis of the SSU rRNA gene determined the prevalence of G. duodenalis (5.81%, 24/413) in diarrheal patients, with a peak in minors aged 5–17 years, and identified assemblages A and B. MLG-AII and MLG-B1 were obtained based on concatenated nucleotide sequences of the bg, tpi, and gdh genes, with MLG-AII being identical to a cat-derived isolate reported previously. By sequence analysis of the SSU rRNA gene, G. duodenalis was detected in 214 (16.14%) animals belonging to 11 mammal species, with the prevalence ranging from 1.69 to 53.85%, and assemblages A to G were identified. Sequence analysis of the bg, tpi, and gdh genes from 46 specimens produced 31 MLGs, including MLG-AI (n = 1), MLG-B2–B8 (n = 18), and MLG-E1–E23 (n = 27). Conclusions: The finding of G. duodenalis in diarrheal patients enhances consciousness of detecting G. duodenalis in clinical practice and emphasizes the importance of health education in local inhabitants, especially in the age group of 5–17 years. The identification of seven assemblages (A to G) and 33 MLGs reveals genetic heterogeneity of G. duodenalis in the investigated areas. Due to insufficient homology data on the zoonotic transmission of G. duodenalis, the precise epidemiological role that animals play in the transmission of human giardiasis needs to be assessed by more large-scale molecular epidemiological investigations of local humans and animals. Graphical

Background: Enterocytozoon bieneusiis the most frequently detected microsporidian species in humans and animals. Currently, to the best of our knowledge, no information onE. bieneusiinfection in Himalayan marmots (Marmota himalayana) and Alashan ground squirrels (Spermophilus alashanicus) is available worldwide. The aim of the present study was to understand the occurrence and genetic characterizations ofE. bieneusiin Himalayan marmots and Alashan ground squirrels in the Qinghai-Tibetan Plateau area (QTPA), Gansu Province, China.Methods: A total of 498 intestinal contents were collected from 399 Himalayan marmots and 99 Alashan ground squirrels in QTPA. These samples were screened for the presence ofE. bieneusiby using nested polymerase chain reaction and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. The ITS-positive sequences were aligned and phylogenetically analyzed to determine the genotypes ofE. bieneusi.Results: The average infection rate ofE. bieneusiwas 10.0% (50/498), with 11.8% (47/399) in Himalayan marmots and 3.0% (3/99) in Alashan ground squirrels. A total of 7 distinctE. bieneusigenotypes were confirmed: 1 known genotype, YAK1 (n = 18) and 6 novel genotypes, named as ZY37 (n = 27), HN39 (n = 1), HN96 (n = 1), SN45 (n = 1), XH47 (n = 1) and ZY83 (n = 1). All the genotypes obtained in the present study were classified into group 1.Conclusions: To our knowledge, this is the first report ofE. bieneusiin Himalayan marmots and Alashan ground squirrels in China. The identification of genotype YAK1 in the two rodent species expanded the host range of this genotype. All the seven genotypes were clustered into zoonotic group 1, suggesting that these animal species can be potential epidemiological vectors of zoonotic microsporidiosis caused byE. bieneusiand pose a threat to ecological security. It is necessary to strengthen management practices and surveillance in the investigated areas to reduce the risk ofE. bieneusiinfection from the two rodent species to humans.

Background: Cryptosporidiumis a highly pathogenic parasite responsible for diarrhea in children worldwide. Here, the epidemiological status and genetic characteristics ofCryptosporidiumin children with or without diarrhea were investigated with tracking of potential sources in Wenzhou City, China.Methods: A total of 1032 children were recruited, 684 of whom had diarrhea and 348 without, from Yuying Children’s Hospital in Wenzhou, China. Samples of stool were collected from each participant, followed by extraction of DNA, genotyping, and molecular identification ofCryptosporidiumspecies and subtypes.Results: Twenty-two of the 1032 (2.1%) children were infected withCryptosporidium spp. with 2.5% (17/684) and 1.4% (5/348) in diarrhoeic and asymptomatic children, respectively. FourCryptosporidiumspecies were identified, includingC. parvum(68.2%; 15/22),C. felis(13.6%; 3/22),C. viatorum(9.1%; 2/22), andC. baileyi(9.1%; 2/22). TwoC. parvumsubtypes named IIdA19G1 (n = 14) and IInA10 (n = 1), and one each ofC. felis(XIXa) andC. viatorum(XVaA3g) subtype was found as well.Conclusions: This is the first research that identifiedCryptosporidiumin children of Wenzhou, China, using PCR. Identification of zoonoticC. parvum, C. felis,C. viatorum, and their subtypes indicate potential cross-species transmission ofCryptosporidiumbetween children and animals. Additionally, the presence ofC. baileyiin children suggests that this species has a wider host range than previously believed and that it possesses the capacity to infect humans.

Intestinal protozoa Cryptosporidium spp., Giardia duodenalis , and Enterocytozoon bieneusi have been implicated in serious waterborne outbreaks worldwide. Wastewater-based epidemiology (WBE) is a promising approach for evaluating the disease prevalence in a catchment population in that it monitors the contamination level of the intestinal pathogens in wastewater. We collected 48 urban wastewater samples (24 from influents and 24 from effluents) from the Yangpu Wastewater Treatment Plant (YPWTP) in Shanghai, China. We identified Cryptosporidium spp., G. duodenalis , and E. bieneusi by nested polymerase chain reaction (PCR) amplification. Cryptosporidium hominis and subtype IdA14 were identified in two samples by analyzing the sequences of small subunit ribosomal RNA (SSU rRNA) and 60–kDa glycoprotein ( gp60 ) genes, respectively. The G. duodenalis sub-assemblage AII ( n = 8) and assemblage C ( n = 4) in 12 samples were determined by analyzing triosephosphate isomerase ( tpi ) gene sequences. The E. bieneusi genotype A was identified in one sample by analyzing the sequence of the internal transcribed spacer (ITS) region of the rRNA gene. These findings suggest that improving wastewater treatment and monitoring the virility of pathogens in effluents is critical. We observed similar prevalence and genotypes/subtypes of the three intestinal protozoa in our wastewater samples as those reported in previous studies, providing evidence that WBE can be used as an effective epidemic management tool. IMPORTANCE: Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common intestinal protozoa causing diarrhea. The infective oocysts, cysts, and spores released in feces can survive in different environments, including multiple types of water bodies. Humans can acquire these intestinal protozoan infections via the fecal-oral route as in waterborne transmission. Wastewater-based epidemiology can rapidly and reliably detect and monitor the emergence and spread of waterborne diseases. We detected Cryptosporidium spp., G. duodenalis, and E. bieneusi in a wastewater treatment plant in Shanghai, China, reflecting the occurrence and genetic characterizations of the three intestinal pathogens from community members served by the wastewater treatment plant.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Introduction: Enterocytozoon bieneusi is the most prevalent microsporidian species infecting humans and causing diarrhea. Epidemiological investigations seldom focus on this pathogen, and its disease burden has been underestimated. This research investigated the prevalence and genotypic characteristics of E. bieneusi in patients with diarrhea from Guangdong (Zhuhai), Shandong (Qingdao), Shanghai and Zhejiang (Wenzhou) Provinces, China. Methods: A total of 691 fecal specimens from patients exhibiting diarrhea were collected and subjected to polymerase chain reaction (PCR) detection, targeting the internal transcribed spacer (ITS) region of the E. bieneusi genome. Genotypes were identified by sequencing PCR products, and zoonotic risk was evaluated through homology and phylogenetic analysis. Results: 4.9% (34/691) of patients were positive for E. bieneusi . The patients from Shanghai had the highest incidence at 14.3% (21/147), which was significantly higher than those in Qingdao (2.9%, 5/171), Wenzhou (3.3%, 3/90), and Zhuhai (1.8%, 5/283) (χ ² = 35.5, P < 0.001). Meanwhile, the positive rate was 7.1% (18/254), 3.1% (10/325) and 5.4% (6/112) among children, adults, and the elderly, respectively. Additionally, the positive rate of patients in rural areas was 8.6% (26/302), significantly higher than that in urban areas, which was 2.1% (8/389) (χ ² = 15.6, P < 0.001). Twelve genotypes of E. bieneusi were identified, including seven known genotypes: CHG3 (n = 17), CHG19 (n = 3), EbPigITS7 (n = 3), Type IV (n = 3), CHG5 (n = 1), EbpA (n = 1), and S7 (n = 1), as well as five novel genotypes (SHH1, WZH1, ZHH1, ZHH2 and ZHH3), each represented by a single sample. The identified genotypes can be categorized into Groups 1, 2, 12, and 13 through phylogenetic analysis. Conclusions: This study offers insights into the epidemiology and genetic diversity of E. bieneusi among patients experiencing diarrhea in four provinces of China. It also underscores the necessity of ongoing monitoring and management to prevent the spread of this pathogen.